One of the earliest events in the formation of a synapse between a motor neuron and a skeletal muscle fiber - a neuromuscular junction - is the aggregation of acetylcholine receptors (AChRs) in the postsynaptic membrane. This step of synapse formation is approximated by rat myotubes maintained in tissue culture, which form large aggregates or clusters of AChRs where they adhere to the tissue culture substrate. The objective of the research proposed here is to learn what macromolecules are responsible for acetylcholine receptor cluster formation. For this purpose rabbit antibodies against known cytoskeletal proteins and mouse monoclonal antibodies against extracts of rat muscle cells will be prepared. These antibodies will be used to determine, by indirect immunofluorescence, if the antigens with which they react are enriched in the myotube regions containing AChR clusters. Two kinds of macromolecules are likely to be associated with these regions: intracellular proteins, perhaps involved in cytoskeletal functions or in binding to AChRs, and extracellular proteins, perhaps involved in adhering the muscle cell to the tissue culture substrate. The antibodies which react with cluster-associated macromolecules will then be used to determine if the same molecules are present at the developing or mature neuromuscular junction. For this purpose, frozen sections through the junctional regions of muscles will be cut and stained by indirect immunofluorescence with the antibodies of interest. The macromolecules so linked to AChR clusters in myotubes and at the neuromuscular junction will be purified by affinity chromatography and characterized using standard biochemical techniques.